Homogeneous persistent luminescence nanocrystals and methods of preparation and application thereof

ABSTRACT

This invention provides a groundbreaking approach to PLNPs and their preparation. In particular, the synthetic methodology disclosed herein fundamentally differs from the traditional solid-state annealing reactions that require extreme and harsh reaction conditions. In one unique aspect of the invention, a simple, one-step mesoporous template method utilizing mesoporous silica nanoparticles (MSNs) is disclosed that affords in vivo rechargeable NIR-emitting mesoporous PLNPs with uniform size and morphology. In another unique aspect of the invention, the novel synthetic approach is based on aqueous-phase chemical reactions conducted in mild conditions, resulting in uniform and homogeneous PLNPs with desired size control (e.g., sub-10 nm).

PRIORITY CLAIMS AND RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/127,376, filed Mar. 3, 2015, the entire content of which isincorporated herein by reference for all purposes.

TECHNICAL FIELDS OF THE INVENTION

The invention generally relates to materials and methods for persistentluminescence. More particularly, the invention relates to uniform andhomogeneous persistent luminescence nanoparticles, and methods ofpreparation and application thereof.

BACKGROUND OF THE INVENTION

Persistent luminescence (PL), also called afterglow or long-lastingphosphorescence, is the phenomenon encountered in materials that glowwell after the end of an excitation with UV or visible light. PLmaterials have been used in a wide variety of applications, such asluminous dials and displays, fiber-optic thermometers, and forensic andmilitary identification materials. While the mechanism underlying PL isnot fully understood, it is generally agreed that the phenomenoninvolves energy traps in a material that are filled during excitation.After excitation, the stored energy is gradually released to emittercenters.

In near infrared (NIR) persistent luminescence, PL phosphors can storeexcitation energy in energy traps while continuing to emit photons forweeks after excitation ceases. (Pan, et al. 2012 Nat Mater 11, 58;Abdukayum, et al. 2013 J Am Chem Soc 135, 14125; Liu, et al. 2013 SciRep-Uk 3, 1554.) Studies have been done to explore advanced biomedicalapplications for NIR persistent luminescence nanoparticles (PLNPs) thatcan emit PL in the optical bio-imaging window (˜650-1000 nm) for hours,or even days, after cessation of excitation. (Pan, et al. 2012 Nat Mater11, 58; Maldiney, et al. 2014 Nat Mater 13, 418.) The temporalseparation of excitation and afterglow properties of these persistentphosphors makes them ideal as in vivo optical imaging contrast reagents.(Chermont, et al. 2007 P Natl Acad Sci USA 104, 9266; Maldiney, et al.2011 J Am Chem Soc 133, 11810; Maldiney, et al. 2011 Acs Nano 5, 854.)Excitation resource and relevant complicated optics that are necessaryfor traditional fluorescence imaging are no longer needed.

Until now, persistent luminescence has relied on short-wavelengthexcitation (e.g., ultraviolet light), which has rather limitedtissue-penetration depth. (Clabau, et al. 2005 Chem Mater 17, 3904; Lin,et al. 2001 J Mater Sci Lett 20, 1505; Rodrigues, et al. 2014 J MaterChem C 2, 1612; Rodrigues, et al. 2012 J Phys Chem C 116, 11232.) Toaddress this problem, a NIR-light-stimulated PL mechanism was proposedin LiGa₅O₈:Cr³⁺, to release energy trapped in deeper energy levels ofthe phosphor. In this case, however, the energy must be pre-charged byUV-light and the photo-stimulated emission continues to weaken aftereach cycle of photo-stimulation and finally becomes extinguished. (Liu,et al. 2013 Sci Rep-Uk 3, 1554; Zhuang, et al. 2013 J Mater Chem C 1,7849.) Very recently, the PL phosphor, ZnGa₂O₄:Cr³⁺ (ZGC), was found tobe activatable using tissue-penetrable red light, which means thatenergy can be recharged and NIR PL imaging is no longer limited by theluminescence-decay life-time of the phosphor. (Maldiney, et al. 2014 NatMater 13, 418.) Thus ZGC is arguably the optimal rechargeable NIRpersistent emitting phosphor reported to date.

Despite such inspiring progress, production of uniformly structured NIRPL ZGC phosphors remains challenging. Advances in the development ofPLNPs for both basic research and commercialization have been hamperedby their complicated synthesis methods. To make such NIR-persistentphosphors bulk crystal requires temperatures greater than 750° C. intraditional solid-state annealing reactions. (Pan, et al. 2012 Nat Mater11, 58; Clabau, et al. 2005 Chem Mater 17, 3904; Setlur, et al. 2008 JAppl Phys 103, 053513.) Moreover, to convert such bulk crystal intonanoparticles that are sufficiently disperse for biologicalapplications, certain tedious physical treatments such as grinding orlaser ablation must be utilized. (Abdukayum, et al. 2013 J Am Chem Soc135, 14125; Liu, et al. 2013 Sci Rep-Uk 3, 1554; Maldiney, et al. 2014Adv Funct Mater DOI 10.1002/adfm.201401612; Maldiney, et al. 2014Nanoscale 6 (22), 13970-13976; Maldiney, et al. 2012 Opt Mater Express2, 261.) The afforded products are generally highly heterogeneous andsuffer from severe agglomeration. In addition, bio-imaging applicationsgenerally require that the nanocrystals be biocompatible, which meansthat the PLNPs need to be comparable in size to the biomolecules theylabel, so as not to interfere with cellular systems.

Thus, novel and improved synthetic methodologies, in particularaqueous-phase chemical synthesis for sub-10 nm NIR PLNPs that areuniform and can be homogeneously dispersed in a carrying medium (e.g.,aqueous solution), are strongly desired.

SUMMARY OF THE INVENTION

The invention provides novel PLNPs that are uniform and homogeneous,aqueous-phase chemical synthesis of such PLNPs, and their applicationsin various fields including biomedical, cosmetics, plastics, inks,security, etc.

In one aspect, the invention generally relates to a method for preparingmesoporous persistent luminescence nanoparticles. The method includesproviding mesoporous silica nanoparticles having mesopores of definedsize and morphology; and reacting precursors to form persistentluminescent nanoparticles templated by the mesopores of the mesoporoussilica nanoparticles under conditions sufficient to form mesoporouspersistent luminescence nanoparticles.

In another aspect, the invention generally relates to mesoporouspersistent luminescence nanoparticles prepared by a method disclosedherein.

In yet another aspect, the invention generally relates to a method forpreparing persistent luminescence nanoparticles. The method includesconducting a hydrothermal chemical reaction in an aqueous phase underconditions sufficient to form uniform and homogeneous persistentluminescence nanoparticles.

In yet another aspect, the invention generally relates to persistentluminescence nanoparticles prepared by a method according to a methoddisclosed herein.

In yet another aspect, the invention generally relates to rechargeablepersistent luminescence nanocomposites comprising doped zinc gallatesZnGa₂O₄:Cr that are substantially uniform and homogeneous with a medianparticle size of less than about 1,000 nm and having specific surfacearea from about 50 m²/g to about 600 m²/g wherein the mesoporouspersistent luminescence nanocomposites is capable of NIR-emitting in therange from about 650 nm to about 900 nm after multiple emission andrecharge cycles (e.g., greater than 5, greater than 10).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Illustration of the synthesis of PL-functionalized MSNs andtheir in vivo imaging application.

FIG. 2. Optimization of the experimental conditions. (A) XRD and (B)photoluminescence spectra (excitation wavelength, 254 nm) of the mZGCsamples synthesized at various temperatures, (C) PL excitation andemission spectra (tested using the phosphorescence mode of thefluorimeter), (D) PL decay curve of mZGC synthesized at 600° C., excitedby UV lamp (254 nm). Sample mass for above measurements is 100 mg.

FIG. 3. Morphologies and porous structure of MSNs and mZGC. FESEM imagesof (A) MSNs and (B) mZGC. (C) TEM and (D) HRTEM images of mZGC, (E) N₂adsorption/desorption isotherms, and (F) pore-size distributions of MSNsand mZGC.

FIG. 4. In situ simulated deep-tissue charging properties of mZGC. (A)Optical image of in situ excitation, (B) PL spectra covered anduncovered by pork layer, (C) in vitro charged and recharged decay curvesof mZGC. All spectra were recorded with the ZGC under an 8-mm porklayer.

FIG. 5. Recharged in vitro PL imaging of mZGC covered by an 8-mm porklayer.

FIG. 6. In vivo recharging of mZGC for PL imaging using a white LED. (A)First charging, (B) 10 min after first charging, (C, E, G, I) second tofifth recharging at time intervals of 10 min, (D, F, H) 10 min aftersecond, third, and forth recharging, (J) background control imaging of amouse without injection of mZGC.

FIG. 7. Biodistribution of mZGC, 2 h after tail-vein injection.

FIG. 8. Optimization of Cr′ doping concentration (vs. Zn) in mZGC.

FIG. 9. Ibuprofen storage/release properties of mZGC. The ibuprofenstorage and release experiment was performed according to a previousreport. Briefly, 60 mg of the MSNs/ZnGa₂O₄:Cr³⁺ were immersed into 1 mLof 20 mg/L ibuprofen cyclohexane solution for 24 h. Excess ibuprofencyclohexane solution was removed by centrifugation and decantation. Thecyclohexane in the mesoporous silica was evaporated in a 50° C. airdryer for 1 h. The in vitro release of ibuprofen was performed byimmersing 60 mg of the drug loaded mZGC in 60 mL of simulated body fluidat 37° C. 100 μL of the mixed solution was taken to test the releasedamount of ibuprofen at fixed time intervals; this was centrifuged todetermine the released ibuprofen in the supernatant by detecting theabsorbance. The initial content of ibuprofen on MSNs and mZGC is 146.3mg/g and 103.9 mg/g respectively, by elemental analysis.

FIG. 10. Cell toxicity of mZGC. The cytotoxicity was tested using3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium bromide (MTT,sigma, USA) assay using human umbilical vein endothelial cells (HUVEcells, ATCC, CRL-1730).

FIG. 11. Spectrum of the LED torch used in experiments. Wavelengthslonger than 600 nm have better biological tissue penetration abilitythan shorter ones, and result in the charging of mZGC in vivo.

FIG. 12. Schematic illustration of the synthesis and imagingapplications of ZGC PLNPs.

FIG. 13. (A) TEM and size distribution, (B) HRTEM, of a single crystal,(C) selected area electron diffraction pattern, and (D) X-raydiffraction analysis of ZGC-1.

FIG. 14. The disperse stability of ZGC-1 and ZGC-2 in water (pH=6.5).(A) Dynamic light scattering patterns of ZGC-1 in water before and afterstorage of 1 month, (B1, B3) bright field and (B2, B4) correspondingluminescence pictures of ZGC-2 in water, (C1, C3) bright field and (C2,C4) corresponding luminescence pictures of ZGC-1 in water. Persistentluminescence intensity is expressed in false color units (1 unit=2.107photons·s-1·cm-2·sr) for all images.

FIG. 15. Decay curves of ZGC-1 excited by various wavelength.

FIG. 16. Superior PL properties of ZGC-1. (A) PL spectra and (B) decaycurves of ZGC-1 and ZGC-2 excited at 650 nm for 200 s using a xenon lampas the light source. PL spectra and decay curves were recorded 30 safter the stop of the excitation. Sample mass=100 mg.

FIG. 17. Analysis of the imaging ability of ZGC-1 and ZGC-2. (A) 50 mgin a black 96-well-plate; (B) in vivo activated imaging aftersubcutaneous injection (50 μL, 2 mg/mL). Persistent luminescenceintensity is expressed in false color units (1 unit=1·10⁶ photonss⁻¹·cm⁻²·sr) for all images.

FIG. 18. Comparative deep tissue imaging of ZGC-1 and ZGC-2. (A and B)deep tissue in vivo imaging before and after in situ excitation; (C-H)repeated imaging before and after 2nd, 3rd, and 4th in situ excitations,time interval=30 min. Excitation with a white LED (5000 lumen) lightsource for 30 s. Persistent luminescence intensity is expressed in falsecolor units (1 unit=4350 photons s⁻¹·cm⁻²·sr) for all images.

FIG. 19. Imaging applications of ZGC PLNPs after ex vivo (A) and in vivo(B) being excited using a white LED light source (5000 lumen).

FIG. 20. Luminescence spectra of PLNPs, mSiO₂/ZnGa₂O₄ doped with diversedopant ions.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a groundbreaking approach to PLNPs and theirpreparation. In particular, the synthetic methodology disclosed hereinfundamentally differs from the traditional solid-state annealingreactions that require extreme and harsh reaction conditions.

In one unique aspect of the invention, a simple, one-step mesoporoustemplate method utilizing mesoporous silica nanoparticles (MSNs) isdisclosed that affords in vivo rechargeable NIR-emitting mesoporousPLNPs with uniform size and morphology.

For example, uniform sized NIR-emitting mesoporous SiO₂/ZnGa₂O₄:Cr³⁺persistent luminescence nanocomposites were made by a simple, one-stepmesoporous template method. The near-infrared persistent luminescence ofthe nanocomposites can be recharged for multiple times both in vitrounder deep tissue simulation (e.g., 8 mm pork slab) and in a live mousemodel. This unconventional approach paves the way for the synthesis andwider application of deep tissue rechargeable ZGC persistentluminescence materials in photonics and biophotonics.

In another unique aspect of the invention, the novel synthetic approachis based on aqueous-phase chemical reactions conducted in mildconditions, resulting in uniform and homogeneous PLNPs with desired sizecontrol (e.g., sub-10 nm). The PLNPs can be homogeneously dispersed in acarrying medium (e.g., aqueous solution).

For example, the aqueous phase synthesis afforded uniformed NIR PL ZGCnanoparticles. The synthesis temperature was significantly decreasedfrom up 1000° C. needed for existing methods to around 250° C., whichgreatly simplifies the reaction equipment. The as-synthesized ZGCnanocrystals can be made as small as a size of 8±4 nm with narrow sizedistribution, which is important for its potential biomedicalapplications. The ZGC nanocrystals can be easily distributed into waterafter a simple acid washing post-treatment, which facilitates itssurface modification of diverse functional group including biomolecules(e.g., antibody). It has been demonstrated that the PLNPs so preparedprovide significantly better PL intensity than those synthesized by thetraditional annealing method and can be activated in deep tissue coveredby 1 cm pork layer, which enables it for various in vivo applications.

In one aspect, the invention generally relates to a method for preparingmesoporous persistent luminescence nanoparticles. The method includesproviding mesoporous silica nanoparticles having mesopores of definedsize and morphology; and reacting precursors to form persistentluminescent nanoparticles templated by the mesopores of the mesoporoussilica nanoparticles under conditions sufficient to form mesoporouspersistent luminescence nanoparticles.

In certain embodiments of the invention, the specific surface area ofthe mesoporous silica nanoparticles is from about 100 m²/g to about2,000 m²/g (e.g., from about 100 m²/g to about 1,000 m²/g, from about100 m²/g to about 800 m²/g, from about 100 m²/g to about 600 m²/g, fromabout 100 m²/g to about 400 m²/g, from about 200 m²/g to about 2,000m²/g, from about 500 m²/g to about 2,000 m²/g, from about 800 m²/g toabout 2,000 m²/g, from about 1,000 m²/g to about 2,000 m²/g).

In certain embodiments of the invention, the mesoporous persistentluminescence nanoparticles are NIR-emitting in the range of about 650 nmto about 900 nm.

In certain embodiments of the invention, the mesoporous persistentluminescence nanoparticles are rechargeable.

In certain embodiments of the invention, the mesoporous persistentluminescence nanoparticles are doped zinc gallates ZnGa₂O₄:Cr.

In certain embodiments of the invention, reacting precursors to formpersistent luminescent nanoparticles is conducted at a temperature in arange from about 400° C. to about 700° C. (e.g., from about 400° C. toabout 650° C., from about 400° C. to about 600° C., from about 400° C.to about 550° C., from about 400° C. to about 500° C., from about 450°C. to about 700° C., from about 500° C. to about 700° C., from about550° C. to about 700° C., from about 600° C. to about 700° C.).

In certain embodiments of the invention, wherein persistent luminescenceis detectable at least 5 hours (e.g., at least 10 hours) afterexcitation.

In certain embodiments of the invention, the formed persistentluminescent nanoparticles are substantially uniform and homogeneous withmedian particle sizes less than about 1,000 nm (e.g., less than about500 nm, less than about 200 nm, less than about 100 nm, less than about50 nm, less than about 20 nm, less than about 10 nm).

In another aspect, the invention generally relates to mesoporouspersistent luminescence nanoparticles prepared by a method disclosedherein.

In yet another aspect, the invention generally relates to a method forpreparing persistent luminescence nanoparticles. The method includesconducting a hydrothermal chemical reaction in an aqueous phase underconditions sufficient to form uniform and homogeneous persistentluminescence nanoparticles.

In certain embodiments of the invention, the hydrothermal chemicalreaction is conducted in aqueous phase at a temperature from about 150°C. to about 300° C. (e.g., from about 150° C. to about 280° C., fromabout 150° C. to about 250° C., from about 150° C. to about 230° C.,from about 150° C. to about 200° C., from about 180° C. to about 300°C., from about 200° C. to about 300° C., from about 250° C. to about300° C.).

In certain embodiments of the invention, the formed persistentluminescent nanoparticles are substantially uniform in particle size ofless than about 10 nm (e.g., less than about 8 nm, less than about 6 nm,less than about 5 nm).

In certain embodiments of the invention, the persistent luminescencenanoparticles are NIR-emitting in the range from about 650 nm to about900 nm (e.g., from about 650 nm to about 850 nm, from about 650 nm toabout 800 nm, from about 650 nm to about 750 nm, from about 750 nm toabout 900 nm, from about 800 nm to about 900 nm).

In certain embodiments of the invention, the mesoporous persistentluminescence nanoparticles are doped zinc gallates ZnGa₂O₄:Cr.

In certain embodiments of the invention, persistent luminescence isdetectable at least 5 hours (e.g., at least 10 hours) after excitation.

In certain embodiments of the invention, the specific surface area ofthe mesoporous silica nanoparticles is from about 100 m²/g to about2,000 m²/g (e.g., from about 100 m²/g to about 1,000 m²/g, from about100 m²/g to about 800 m²/g, from about 100 m²/g to about 600 m²/g, fromabout 100 m²/g to about 400 m²/g, from about 200 m²/g to about 2,000m²/g, from about 500 m²/g to about 2,000 m²/g, from about 800 m²/g toabout 2,000 m²/g, from about 1,000 m²/g to about 2,000 m²/g).

In yet another aspect, the invention generally relates to persistentluminescence nanoparticles prepared by a method according to a methoddisclosed herein.

In yet another aspect, the invention generally relates to rechargeablepersistent luminescence nanocomposites comprising doped zinc gallatesZnGa₂O₄:Cr that are substantially uniform and homogeneous with a medianparticle size of less than about 1,000 nm and having specific surfacearea from about 50 m²/g to about 600 m²/g wherein the mesoporouspersistent luminescence nanocomposites is capable of NIR-emitting in therange from about 650 nm to about 900 nm after multiple emission andrecharge cycles (e.g., greater than 5, greater than 10).

In certain embodiments of the invention, the rechargeable persistentluminescence nanocomposites are rechargeable in vivo bytissue-penetrable red excitation.

In certain embodiments of the invention, the specific surface area ofthe mesoporous silica nanoparticles is from about 50 m²/g to about 600m²/g (e.g., from about 50 m²/g to about 500 m²/g, from about 50 m²/g toabout 300 m²/g, from about 50 m²/g to about 200 m²/g, from about 50 m²/gto about 100 m²/g, from about 100 m²/g to about 600 m²/g, from about 200m²/g to about 600 m²/g, from about 300 m²/g to about 600 m²/g, fromabout 400 m²/g to about 600 m²/g).

In certain embodiments of the invention, the median particle sizes areless than about 1,000 nm (e.g., less than about 500 nm, less than about200 nm, less than about 100 nm, less than about 50 nm, less than about20 nm, less than about 10 nm).

In certain embodiments of the invention, the persistent luminescencenanocomposites can emit multiple colors (2, 3, 4 or more colors).

In certain embodiments of the invention, the persistent luminescencenanocomposites are components of an imaging or diagnostic probe or alabeling agent for biomedical assays (e.g., western blot).

In certain embodiments of the invention, the persistent luminescencenanocomposites are components of a drug delivery vehicle or atherapeutic agent.

In certain embodiments of the invention, the persistent luminescencenanocomposites are components of a nail polish.

In certain embodiments of the invention, the persistent luminescencenanocomposites are components of a hair spray.

In certain embodiments of the invention, the persistent luminescencenanocomposites are components of ink for latent fingermark, and for artillustration and paints, tracer in dark conditions such as at night andsubmarine condition, for constructions, living goods and utility device.

In certain embodiments of the invention, the rechargeable persistentluminescence nanocomposites are homogenously dispersed in a solution orsuspension of water or an organic solvent.

I. One-Step Templated Synthesis of NIR-Emitting Mesoporous PLNPS

Because of their readily controllable synthesis and resultingmorphology, super-high specific surface area, huge pore volume, and goodbiocompatibility, MSNs are widely utilized in biology, drug delivery,and medicinal applications to encase various functionalmolecules/luminescence contrast reagents. (Yamashita, et al. 2013Advanced Drug Delivery Reviews 65, 139; Liu, et al. 2013 Angew Chem IntEdit 52, 4375; Fan, et al. 2014 Biomaterials 35, 8992.)

As first disclosed herein, MSNs can be used to template the synthesis ofZGC NIR persistent phosphors in situ with defined size and morphology(mZGC). Since temperatures beyond 750° C. may lead to the collapse ofthe mesoporous silica nanostructures, the reaction temperature for thephosphor synthesis was explored systematically in this study. It wasfound that at 600° C., the as-synthesized mZGC preserves defined size,morphology and mesoporous nanostructure of the MSNs as well aspossessing optimal luminescence properties. Further, the performance ofmZGC in imaging was measured both in vitro and in vivo to assesspotential applications in biophotonics. It was demonstrated thatas-synthesized mZGC can be recharged in a simulated deep-tissueenvironment (˜8 mm pork slab) in vitro using red light. Moreover, it wasobserved that mZGC can be repeatedly activated in vivo for persistentluminescence imaging in a live mouse model using white LED as a lightsource.

Formation of mZGC Using MSNs as Nanoreactors

The MSNs were impregnated with ZGC nitrate precursor solutions, whichentered the nanochannels of MSNs with ease due to the capillarity of themesopores. The optimal used composition was determined atZn/Ga/Cr=1/1.997/0.003 by molar ratio (FIG. 8). ZGC was formed in thenanochannels of the MSNs after vacuum drying and annealing, as shown inFIG. 1. By measuring the mass of the used silica and the total mass ofthe as-formed nanocomposites, the content of ZGC in mZGC was calculatedto be 10.4% by weight. (Table 1). Such a simple, one-step mesoporoustemplate method allowed preparation of NIR-persistent-luminescentmesoporous nanocomposites that combine the unique optical properties ofNIR persistent phosphors and the mesoporous attributes of mesoporoussilica.

TABLE 1 Test of the ZGC content in as-synthesized mZGC product IncreasedZGC content in MSNs mZGC mass mZGC (by weight) 400.0 mg 445.7 mg 45.7 mg10.4% ± 0.4% 400.0 mg 448.1 mg 48.1 mg 400.0 mg 445.4 mg 45.4 mg

TABLE 2 Mass loss of mZGC after rinsed in PBS for 48 hours Rinsed anddried decreased Mass loss mZGC mZGC mass by weight 100.0 mg  99.7 mg−1.7 mg 0.2% ± 1.5% 100.0 mg 101.4 mg +1.4 mg 100.0 mg 100.1 mg +0.1 mg

X-ray diffraction (XRD) and luminescence measurements were used toconfirm the nature of the as-synthesized nanocomposite. The XRD results(FIG. 2A) showed that the diffraction characteristics (peaks at 30.4°,35.78°, 43.50°, 57.48°, 63.12°) of the ZGC crystal already appear whenthe annealing temperature reaches 500° C., which then become clear whenthe temperature increases to 600° C. This is in agreement with thepresence of the spinel phase ZnGa₂O₄ (JCPDS index no. 01-086-0410).However, impurity peaks start to appear at 700° C., which indicate theformation of metal silicates (arrows in FIG. 2A). As a result, it wasobserved that the PL intensity (FIG. 2B; 650-750 nm) initially increaseswith temperature (from 400-600° C.) and reaches the optimal intensity at600° C. before decreasing at 700° C.

The phosphorescence excitation spectrum of the sample synthesized at600° C. consists of three ZGC characteristic excitation bands (FIG. 2C).These bands, which range from <350 nm, 350-470, and 470-650 nm, can beattributed to the band-to-band transitions of ZnGa₂O₄, ⁴A₂→⁴T₁, and the⁴A₂→⁴T₂ transition of Cr³⁺, respectively. (Bessiere, et al. 2011 OptExpress 19, 10131.) Among these bands, the absorbance at 600-650 nm isresponsible for the in vivo recharging using deeper-tissue-penetratingred light. (Maldiney, et al. 2014 Nat Mater 13, 418.) The PL spectrum(FIG. 2C) of the mZGC, which is around 696 nm (inside the NIR imagingwindow, which ranges from 650-900 nm), is also consistent with theresults found in the traditional solid-state reaction. (Bessiere, et al.2011 Opt Express 19, 10131; Zhuang, et al. 2013 Appl Phys Express 6.052602; Zhuang, et al. 2014 J Mater Chem C 2, 5502.) The PL of ZGC canbe detected even after more than 5 h followed by an excitation for 5 minwith a UV light source (254 nm; FIG. 2D). Synthesis of zinc gallatebased phosphors can usually only occur at temperatures at least 750° C.(Bessiere, et al. 2011 Opt Express 19, 10131; Zhuang, et al. 2013 ApplPhys Express 6. 052602; Allix, et al. 2013 Chem Mater 25, 1600.)

The decrease in synthesis temperatures from those generally used impliesthat the utilization of the pores of MSNs as nanoreactors facilitatesthe formation of ZGC phosphor. (A. R. West, Solid-State Chemistry andIts Applications (second edition), John Wiley & Sons, Ltd, 2014, 187.)Homogeneous distribution of these reactants in confined mesoporousnanostructures results in a higher reactivity than would otherwise bethe case; such phenomena were observed previously. (Zhan-Jun, et al.2012 J Mater Chem 22, 24713; Chen, et al. 2014 Adv Mater 26, 4947; Li,et al. 2014 Opt Express 22, 10509; Li, et al. 2014 Green Chem 16, 2680.)

Morphology and Porous Structure of mZGC

This substantial decrease in synthesis temperature is of vitalimportance to generate mZGC nanocomposites successfully since thenanochannels of MSNs start to collapse when annealed at temperatureshigher than 600° C. (Li, et al. 2013 Micropor Mesopor Mat 176, 48.) Thefield-emission scanning electron microscopy (FESEM) images of theas-synthesized MSNs (FIG. 3A) and the corresponding mZGC (FIG. 3B)indicate that the MSNs survive calcination at 600° C. No apparentmorphological change could be observed. Furthermore, in the transmissionelectron microscopy (TEM) and high-resolution-TEM (HRTEM) images (FIG.3C, 3D), tiny dark spots (ZGC nanoparticles) appear homogeneously in thenanochannels of the MSNs.

N₂ adsorption/desorption was performed to study the influence of thesynthesis process on the specific surface area and pore-sizedistribution of the MSNs. Apparent mesoporous characteristics of plateauregions can be observed both for the MSN templates and for theas-synthesized mZGC (FIG. 3E). The specific surface area of the MSNsaccording to the Brunauer-Emmett-Teller (BET) method decreased from554.2 m²/g to 214.6 m²/g, while the pore volume decreased from 0.3395cm³/g to 0.1562 cm³/g, which might arise from the formation of particlesin the mesopores. The synthesis of mZGC from the MSNs did not decreasethe overall average pore size of the carriers. In fact, a slightincrease in average pore size from 2.450 nm to 2.912 nm was observed, asshown in FIG. 3F, which may be explained by the ZGC existing asisolated, tiny particles in the nanochannels of the MSNs, and this isalso evidenced by the HRTEM image. The mesoporous properties of theas-synthesized mZGC were also verified by the sustained release of awidely used model cargo, ibuprofen (FIG. 9). Thus, the PL nano-carrierapproach disclosed herein synergized both unique optical properties ofZGC and the cargo storage/release properties of MSNs.

Imaging Capability of mZGC Through Simulated Deep Tissue

Most of the current PL phosphors can only be excited effectively underblue or even UV light, which can hardly penetrate the deep tissue ofanimals. (Pan, et al. 2012 Nat Mater 11, 58; Chermont, et al. 2007 PNatl Acad Sci USA 104, 9266; Clabau, et al. 2005 Chem Mater 17, 3904.)The PL excitation band from 600-650 nm is in the transmission window ofbiological tissue (600-1100 nm) and thus gives us the opportunity torecharge the energy-exhausted mZGC in deep tissue in situ. (Maldiney, etal. 2014 Nat Mater 13, 418.) Since there is no standard method toaccurately evaluate the deep-tissue-imaging ability of PL phosphors, ameat-covering method is proposed for comparison according to ourprevious report on up-conversion imaging (FIG. 3A). (Chen, et al. 2012Acs Nano 6, 8280.) The as-synthesized mZGC sample disk could be excitedat 620 nm when covered by 8 mm of pork. After switching off the lightused for excitation, the PL spectrum can recorded using afluorospectrometer (FluoroMax-3, HORIBA, USA) fitted with aphotomultiplier tube (PMT) detector. A similar PL spectrum can beobtained to that of the uncovered sample, which means that the NIR PL ofmZGC can efficiently penetrate pork tissue as thick as 8 mm, as shown inFIG. 4B. The in situ excited PL of mZGC could be reproducedconsecutively in situ more than five times on any occasion, as shown inFIG. 4C.

With the demonstration that the PL of mZGC can be charged by red lightunder an 8 mm pork layer, for the first time its applications arestudies in deep-tissue imaging using a white light-emitting diode (LED)as the excitation light source (spectrum shown in FIG. 11). Therechargeable PL imaging of the mZGC sample was performed five timesunder a pork layer of 8 mm without any obvious signal weakening, whichimplies its potential for use in in vivo imaging applications (FIG. 5).For in vivo applications, good biocompatibility is anticipated since theNIR PL phosphor, ZGC, is mainly incorporated within the mesopores of theMSNs, which are well-known to be biocompatible. No apparent cellulartoxicity were observed for mZGC under an exposure concentration as highas 50 mg/L (FIG. 10).

To study the in vivo chargeability of mZGC, the mZGC saline solution(200 μL, 5 mg/mL) was injected into a live mouse through the tail vein.After exposure to white LED light, a satisfactory PL imaging picture wasobtained, as shown in FIG. 6. Moreover, after re-performing the in situexcitation, the PL of ZGC was recharged again. No apparent decrease inPL signal was observed after five imaging/recharging cycles (FIG. 6). Ahigh signal-to-noise ratio of ˜40:1 was obtained by comparing the PLsignal from liver area of mouse with and without injecting mZGC. Thebiodistribution of the mZGC was studied after euthanasia of the mouse (2h after injection). Most of the PL signals emanate from the liver andspleen, which is consistent with the in vivo imaging results (FIG. 7).Given the in vivo chargeable PL attributes, the detection of the PLfunctionalized mesoporous carriers in vivo is not limited by the decayof the PL intensity. Thus, the as-synthesized mZGC has great potentialto become a new class of mesoporous nanocomposites with NIR PLproperties.

Thus, disclosed herein is an in vivo rechargeable NIR-emittingmesoporous SiO₂/ZnGa₂O₄:Cr³⁺ PL nanocomposites. By using the mesoporesof MSNs as a reaction template, the synthesis temperature of thepersistent phosphor, ZnGa₂O₄:Cr³⁺ was decreased from higher than 750° C.in a solid-state reaction to only 600° C. At this lower temperature,both the unique mesoporous attributes, and the uniform size andmorphology of the MSNs were retained in the nanocomposites. For thefirst time, it was confirmed that mZGC could be repeatedly charged insitu under a deep-tissue layer of 8 mm. The deep-tissue chargeable PLproperties of mZGC also ensured its repeatable recharged PL imaging in alive mouse model. It is worth noting that this observation is the firstdirect evidence that persistent luminescence can be recharged in vivofor multiple times. This concept of utilizing mesoporous silica asnanoreactor to fabricate ZGC PL nanoparticles with uniform morphologyand preserved porous nanostructure will be significant in directing thesynthesis of mesoporous PL systems with diverse PL phosphors and paves anew way to the wide application of deep tissue rechargable ZGC inphotonics and biophotonics.

Experimental Materials

Tetraethoxysilane (TEOS), ethanol, diethanolamine (DEA), ammoniumhydrate, cetyltrimethylammonium bromide (CTAB), Ga₂O₃, Zn(NO₃)₂.6H₂O,Cr(NO₃)₃.9H₂O, and concentrated nitric acid were all of analyticalstandards, purchased from Sigma-Aldrich and were used as-received.Ga(NO₃)₃ solution was prepared by dissolving Ga₂O₃ in 1:1 concentratednitric acid followed by air drying at 105° C. to remove excess nitricacid, and then redissolving in deionized water.

Synthesis of MSNs

The MSN synthesis was modified from that in a previous report.(Zhan-Jun, et al. 2012 J Mater Chem 22, 24713.) Briefly, 7 mL ofethanol, 0.2 g of CTAB, and 50 μL of diethanolamine were dissolved in 25mL of water under stirring at 60° C. for 30 min to prepare a transparentsolution. Then, 2 mL of tetraethoxysilane were added rapidly. Thereaction was finished after stirring for another 2 h. Mesoporous silicananospheres (about 100-nm diameter) were collected by centrifugation andcalcination at 550° C. for 2 h to remove CTAB and possible organicresidues.

Synthesis of mZGC Nanocomposites

A precursor solution was prepared by dissolving the correspondingnitrates in water/ethanol (1/1, v/v). The final concentration of Zn²⁺,Ga³⁺, and Cr³⁺ was controlled to be 0.5 mol/L, 0.9985 mol/L, and 0.0015mol/L, according to a stoichiometric ratio of Zn/Ga/Cr (1/1.997/0.003,molar ratio), respectively. 200 μL of the precursor solution were mixedwith 200 mg mesoporous silica and the mixture was dried in a vacuum ovenat 50° C. for 12 h. The samples were then put into a muffle furnace andthe temperature was slowly increased by 5° C./min.

In Vitro and In Vivo PL Imaging of mZGC

In vitro imaging was performed by putting the powder (100 mg) sampleinto a black 96-well plate covered with an 8-mm layer of pork tissue.The PL signal from the covered mZGC was recorded after illuminationusing an LED (5000 lumen) for 15 s. The in vivo imaging was conducted byinjection of the mZGC dispersion in phosphate-buffered saline (PBS; 5mg/mL) through the tail vein. The sample was stored in a dark box for 1day before injection to ensure no pre-activation occurred.

Characterization

X-ray powder diffraction (XRD) measurements were performed on adiffractometer equipped with Cu Kα radiation (λ=1.5418 Å) (PanalyticalX′pert PRO, The Netherlands). The morphology of the samples wasinspected using field-emission scanning electron microscopy (FESEM,HITACHI S-4800, Japan) and transmission electron microscopy (TEM,HITACHI H-7650, Japan) at accelerating voltages of 5 and 100 kv,respectively. High-resolution TEM (HRTEM) images were recorded using aJEM-1200EX II transmission electron microscope. N₂ adsorption/desorptionisotherms were obtained on a full-automatic physical and chemicaladsorption apparatus (micromeritics, ASAP2020C, USA). Pore sizedistribution was calculated from the adsorption branch of N₂adsorption/desorption isotherm and the Brunauer-Emmett-Teller (BET)method. The BET specific surface areas were calculated using the databetween 0.05 and 0.35 just before the capillary condensation. The totalpore volumes were obtained by the t-plot method. Total organic carbonanalyzer was used to determine the exact loading level of ibuprofen onmZGC using a CNS elemental analyzer (Elementar Portfolio, Vario MAX,Germany). The spectra and lifetimes of the samples were tested usingpowdered samples. The photoluminescence spectra and time-decay curveswere measured using a fluorospectrophotometer (FluoroMax-3, HORIBA,USA). The absorbance was detected on a UV-vis spectrometer (Thermo,Evolution300, USA). The PL imaging of mZGC was conducted in a XenogenIVIS imaging system.

II. Facile Solution-Phase Chemical Synthesis of NIR PersistentLuminescence Nanocrystals

Hydrothermal synthesis refers to the synthesis by chemical reactions ofsubstances in a sealed heated solution above ambient temperature andpressure. This technique is considered to be one of the widely usedtechnologies to produce nanocrystals (Shi, et al. 2013 Chem Soc Rev 42,5714.)

Existing fabrication of NIR ZGC PLNPs must undergo solid-state-annealingmethod at extreme high temperatures and complicated physical methodshave to be used to convert as-synthesized large bulk crystals intonanosized particles. As the result, these particles are usuallyheterogeneous, relative larger, and form agglomerates fast in solution.In addition, a key requirement for bioimaging applications is that thenanocrystals be biocompatible, which means that they need to becomparable in size to the biomolecules they are labeling, so as not tointerfere with cellular systems. To date, however, there has been noreport of direct aqueous-phase chemical synthesis of NIR PL materials,not to mention sub-10 nm nanoparticles.

As disclosed herein, water-soluble ZnGa₂O₄Cr_(0.004)PLNPs, withsub-10-nm size and intense PL properties were directly synthesized inaqueous solution by hydrothermal method. An initially attempt was toprepare PLNPs by dissolving Zn(NO₃)₂, Ga(NO₃)₃, and Cr(NO₃)₃ precursorsolutions in a sealed Teflon-lined autoclave and underwent ahydrothermal processing at 250° C. for 10 h (FIG. 12). ZGC PLNPs weregenerated with a narrow size distribution and an average size of 8±4 nm(denoted as ZGC-1; FIG. 13A). The ultra-small size was further confirmedby analysis of the full width at half maximum values of the XRDdiffraction peaks and by the Schërrer equation, with an averagecalculated particle size of 8.7±2.4 nm. As shown in FIG. 13B,as-synthesized ZGC-1 has a clear lattice fingerprint of a (311) plane ofcubic ZnGa2O4. The selected area's electron diffraction pattern (SAED)result, shown in FIG. 13C, indicates that the ZGC nanoparticles have aclear crystal structure and that all of the diffraction circles can beattributed to cubic ZnGa2O4. The formation of pure ZnGa2O4 was alsoconfirmed by analysis of its XRD pattern to that of standard data (JCPDScard, no. 01-082-0466; FIG. 13D).

A stable dispersed colloidal solution is usually a premise for thesurface modification of nanoparticles. Unfortunately, the re-dispersionof ZGC synthesized by conventional solid state annealing reaction(ZGC-2) is quite difficult due to the severe agglomeration caused byhigh temperatures. In contrast, the as-synthesized ZGC-1 can bere-dispersed (or “dissolved”) easily in water forming a transparentcolloidal solution after a simple acid treatment. Dynamic lightscattering analysis was used to analyze the storing stability of ZGC-1.No significant change was observed in the hydrodynamic size distributionof ZGC-1 colloid even after storing for one month (FIG. 14A). Incontrast to the as-synthesized ZGC-1, apparent precipitation wereobserved 30 minutes after milling and ultrasonic treatment of the ZGCdispersion, which was synthesized according to high temperatureannealing method (ZGC-2) (FIGS. 14B and 14C).

Further systematically studied were the PL properties of ZGC-1 underdiverse excitation wavelengths ranging 500-650 nm using the xenon lampin a fluorimeter as the light source without any corrections made (FIG.15). The results indicated that the PL decay curves of the ZGC-1 can beexcited by wavelengths as far as 650 nm. Importantly, during fiverepeated excitation and decay cycles at 650 nm, it was found that the PLintensity of ZGC-1 was nearly double relative to ZGC-2 30 s after thestop of the excitation (FIG. 16).

A comparison imaging experiment was performed both in vitro and in vivousing a commercial white LED (5000 lumen, CREE-T6) as light source. Byapplying the ROI tools of the imaging device, the charging ability ofZGC-1 vs ZGC-2 was compared. The in vitro imaging results indicate anincrease of PL intensity of ZGC-1 by a factor of 2.7, as shown in FIG.17A. While the in vivo results indicate a factor of 2.0 for ZGC-1, asshown in FIG. 17B. Simulated deep-tissue imaging was further performedwith live mouse models covered by a pork slab ˜1 cm thick. The PL ofZGC-2 could hardly be observed under our simulated deep-tissueenvironment, whereas ZGC-1 still provided clear, quality images (FIGS.18A and 18B). The PL signal is eliminated completely 30 min afterexcitation without influencing the next imaging cycle. Once again, itcan be reactivated at a desired time to provide PL images (FIGS.18C-18H).

Based on our observations, ZGC-1 is more suited to deep-tissue imagingapplications. The in vivo imaging performance of ZGC-H was alsoperformed using a live mouse model (FIG. 19). The PL of ZGC-1 could beapplied to track its bio-distribution in live mouse. After the ex vivocharged PL was exhausted, the PL emission could be recharged in vivo byillumination the mouse with a white LED. Although the intensity isrelatively weak than that excited ex vivo, the total traceable time wasnot limited by the decay attribute of persistent phosphor. Moreimportantly, it was found that the ZGC-1 PLNPs can be in vivo activatedthrough bony structure after being injected inside the spinal tube. ThePL imaging result in the live mouse model is consistent with the oneperformed after open the spinal bone, indicating potential applicationsin the central neural system or any imaging application through thebone.

Experimental Materials

Ga₂O₃, Zn(NO₃)₂.6H₂O, Cr(NO₃)₃.9H₂O, ammonium hydroxide, hydrochlorideacid, and concentrated nitric acid were all analytical reagents and usedas received. Ga(NO₃)₃ solution was prepared by dissolving Ga₂O₃ in 1:1concentrated nitric acid followed by air drying at 105° C. to remove theexcess amount of nitric acid and re-dissolved in deionized water. 1mol/L Zn(NO₃)₂, 2 mol/L Ga(NO₃)₃, 4 mmol/L Cr(NO₃)₃ were stored asprecursor solution. A little nitric acid was used to prevent hydrolysisof Ga(NO₃)₃. Polyethyleneimine (PEI) and bovine serum albumin (BSA) werepurchased from Sigma-Aldrich and used directly without furtherpurification.

Synthesis of ZGC-1 by Hydrothermal Process

A certain amount of Ga(NO₃)₃ (2 M), Zn(NO₃)₂ (1 M), Cr(NO₃)₃ (4 mM)precursor solution were mixed with predetermined molar ratio to form aprecursor solution. Ammonium hydroxide (28%, wt) was added quickly undervigorous stirring to reach a pH of 9. The metal hydroxide precursor wassealed into a Teflon-lined autoclave and then performed hydrothermalprocess at 220° C. for 10 hours. ZGC nanocrystals can be obtained withina wide total concentration molar ratio of precursor, which influencedthe product sizes and distributions. In a typical procedure, 2 mmol ofZn(NO₃)₂, 2 mmol of Ga(NO₃)₃, 0.004 mmol Cr(NO₃)₃ were mixed togetherunder vigorous stirring. The total volume was adjusted to 15 mL byadding deionized water. Concentrated ammonium hydroxide (28%) solution(about 1 mL) was added rapidly to adjust the pH to be 9-9.5. Whiteprecipitate was formed immediately. After further stirring for half anhour, the mixture were transmitted into a Teflon-lined autoclave (25 mL)and sealed. The autoclave was put in an oven at 220° C. for 10 hour andthen cooled to room temperature naturally. The white precipitate couldbe obtained easily by centrifugation. The product could be dispersed in0.01 M HCl forming transparent solution. Possible ZnO impurity could beremoved by acid washing. ZGC nanocrystals could be obtained bycentrifugation after diluted with excess isopropanol. Finally, ZGCnanocrystals were redispersed in deionized (DI) water for furthercharacterization and imaging application. The storing concentration ofZGC is approximately 4 mg/mL and used for further modification.

For the dispersion in cell culture medium, 200 μL ZGC-1 storage solutionwas first diluted with 1 mL water and then added into 5 mL of cellculture medium drop by drop under stirring. A clear solution can beobtained after 10 min ultrasonic dispersion.

PEI could be adsorbed to the crystal surface of ZGC-1 due to theelectrostatic interaction with the negatively charged ZGC-1nanocrystals, forming PEI modified ZGC (ZGC/PEI). Briefly, 1 mL of ZGC-1storing solution was added into 10 mL 1 mM of HCl solution, and stirringfor 30 min. 1 mL of PEI solution (20%, wt) was then added rapidly undervigorous stirring. ZGC-PEI could be collected by centrifugation afterdiluted with excess isopropanol. ZGC-PEI was redispersed in DI waterunder ultrasonic dispersion.

ZGC-1 was also modified with BSA. 1 mL of ZGC-1 solution (4 mg/mL) wasadded into 10 mL of BSA solution (1% by weight) drop by drop understirring followed by further stirring for another 30 min. BSA modifiedZGC-1 (ZGC-BSA) was collected by centrifugation and redispersed in 10 mLof DI water by ultrasonic dispersion.

Synthesis of ZGC-2 by Hydrothermal Assisted Solid State Annealing Method

GC-2 was synthesized for comparison according to reported hydrothermalassisted annealing method. 1 mmol of Zn(NO₃)₂, 2 mmol of Ga(NO₃)₃, 0.004mmol Cr(NO₃)₃ were mixed together under vigorous stirring. The totalvolume was adjusted to 10 mL by adding deionized water. Concentratedammonium hydroxide (28%) solution (about 1 mL) was added rapidly toadjust the pH to 7.5. The mixture was sealed into a Teflon-linedautoclave at 120° C. for 24 hour. The resulting precipitate has no PLproperties and was sintered at 750° C. for 5 hours. A small amount of 5mM NaOH solution was added to the powder product to form a slurryfollowed by wet grinding for 1 hour with a mortar and pestle. Theproduct was washed by ethanol (90%) and then dried at 60° C.

Synthesis of ZGC-3 by Using Hydrothermal Post-Treatment

The as-synthesized ZGC-2 was mixed with ammonium aqueous solution (pH9˜9.5) and sealed in an autoclave, which underwent a hydrothermalprocedure at 220° C. for 10 hours. The as-synthesized ZGC-3 wascentrifuged and dried at 80° C.

Imaging

A white LED (5000 lumen, CREE-T6) was used for all the imagingexperiments as the light source. For the in vitro experiment, 50 mg ofZGC sample was put in one well of a black 96-well-plate. The plate wasexposed to the LED for 30 s and then put into the IVIS imaging system todetect the PL signal. The in vivo imaging was performed aftersubcutaneous injection of the sample dispersion (50 μL, 2 mg/mL) and 30s of in situ LED excitation. The in vivo deep tissue recharged PL of ZGCwas performed by covering the subcutaneous injection site with a 1-cmpork slab. The imaging was performed immediately after 30 s of in situLED excitation.

Thus, a novel aqueous phase-based synthetic methodology has now beendemonstrated for the preparation of uniformed NIR PL ZGC nanoparticlesthat can be homogenously dispersed in an aqueous solution or suspension.The synthesis temperature was significantly decreased from up 1000° C.to 250° C., which greatly simplifies the reaction equipment. Theas-synthesized ZGC nanocrystals can be made as small as a size of 8±4 nmwith narrow size distribution, which is important for its potentialbiomedical applications (FIG. 13). The ZGC-1 product can be easilydispersed into water after a simple acid washing post-treatment, whichwill facilitate its surface modification of diverse functional groupincluding biomolecules (e.g., antibody)(FIG. 14). The ZGC-1 product canbe activated in deep tissue covered by 1 cm pork layer, which willfacilitate its applications in animal models (FIG. 18). The ZGC-1 hasshown significantly superior PL intensity than that synthesized usingtraditional annealing method (FIG. 16-18).

The luminescence color of the PLNPs can be further tuned by altering thedopant ions, such as, Gd³⁺ (blue), Bi³⁺ (white), Mn²⁺ (orange) and Cr³⁺(near-infrared). Due to the uniform morphologies and ultra-small sizes,persistent luminescence nanoparticles disclosed herein can be easilydispersed into various solvents, such as water, ethanol, ethyl acetate.

LNPs disclosed herein may be used in biomedical application. The NIRpersistent luminescence mZGC may be developed as an in vivo traceabledrug delivery system. PLNPs can also be used in tumor imaging. As noexcitation will be needed during the imaging procedure, high qualityimaging result can be obtained by removal of the bio-autofluorescencecaused by excitation. mZGC disclosed herein can not only provide tumorimaging ability but also can carry load of functional molecules such asgene, protein, theronostic reagents.

LNPs may be applied in versatile applications in addition to their vastutilities in the healthcare and medical research. For example, they canbe dispersed easily into solutions such as nitrocellulose ethyl acetatesolution, which is the main content of most commercial nail polish.Also, the PLNPs can be dispersed in polyvinyl acetate/ethanol solution,which is a common solvent used in hair spray. The addition of persistentluminescence nanoparticles in nail polish or hair spray will formcolorful afterglow emissions. At night or wherein the light is dark, theafterglow emission can form cool, beautiful nails or hair. Similarapplications may also be found in shoe polish, hair spray, art pigment,etc.

isible persistent luminescence materials have found application asafterglow fingermark powders used with the visualization of latentfingermarks deposited on multicolored substrate surfaces that canpresent a contrast problem if developed with regular fingermark powders.Persistent phosphors can provide a strong afterglow effect which aid inthe establishment of better fingermark detection. Regard the NIRpersistent phosphor, such as our synthesized mZGC, the mZGC disclosedherein can not only image the latent fingermark but also be utilized ininformation encryption. If mZGC is printed on papers, the NIR persistentluminescence cannot be seen by naked eyes. The NIR luminescence can onlybe visualized by using special NIR imaging instrument. Thus, NIRpersistent luminescence has great potential in the field of informationencryption and counterfeit labels.

In this specification and the appended claims, the singular forms “a,”“an,” and “the” include plural reference, unless the context clearlydictates otherwise.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. Although any methods and materials similar or equivalent tothose described herein can also be used in the practice or testing ofthe present disclosure, the preferred methods and materials are nowdescribed. Methods recited herein may be carried out in any order thatis logically possible, in addition to a particular order disclosed.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made in this disclosure. All such documents arehereby incorporated herein by reference in their entirety for allpurposes. Any material, or portion thereof, that is said to beincorporated by reference herein, but which conflicts with existingdefinitions, statements, or other disclosure material explicitly setforth herein is only incorporated to the extent that no conflict arisesbetween that incorporated material and the present disclosure material.In the event of a conflict, the conflict is to be resolved in favor ofthe present disclosure as the preferred disclosure.

EQUIVALENTS

The representative examples are intended to help illustrate theinvention, and are not intended to, nor should they be construed to,limit the scope of the invention. Indeed, various modifications of theinvention and many further embodiments thereof, in addition to thoseshown and described herein, will become apparent to those skilled in theart from the full contents of this document, including the examples andthe references to the scientific and patent literature included herein.The examples contain important additional information, exemplificationand guidance that can be adapted to the practice of this invention inits various embodiments and equivalents thereof.

What is claimed is: 1-16. (canceled)
 17. Rechargeable persistentluminescence nanocomposites comprising doped zinc gallates ZnGa₂O₄:Crthat are substantially uniform and homogeneous in particle sizes of lessthan about 1000 nm and have specific surface area from about 50 m²/g toabout 600 m²/g, wherein the mesoporous persistent luminescencenanocomposites are capable of NIR-emitting in the range of 650 nm and900 nm after multiple emission and recharge cycles.
 18. The rechargeablepersistent luminescence nanocomposites of claim 17, being rechargeablein vivo by tissue-penetrable red excitation light.
 19. The rechargeablepersistent luminescence nanocomposites of claim 18, capable of emittingmultiple colored lights.
 20. The rechargeable persistent luminescencenanocomposites of claim 17, wherein the persistent luminescencenanocomposites are components of an imaging or diagnostic sensing probeor a labeling agent for a biomedical assay.
 21. The rechargeablepersistent luminescence nanocomposites of claim 17, wherein thepersistent luminescence nanocomposites are components of a drug deliveryvehicle.
 22. The rechargeable persistent luminescence nanocomposites ofclaim 17, wherein the persistent luminescence nanocomposites arecomponents of a therapeutic agent.
 23. The rechargeable persistentluminescence nanocomposites of claim 17, wherein the persistentluminescence nanocomposites are components of a nail polish.
 24. Therechargeable persistent luminescence nanocomposites of claim 17, whereinthe persistent luminescence nanocomposites are components of a hairspray.
 25. The rechargeable persistent luminescence nanocomposites ofclaim 17, wherein the persistent luminescence nanocomposites arecomponents of a latent fingermark in ink.
 26. The rechargeablepersistent luminescence nanocomposites of claim 17, being homogenouslydispersed in a solution or suspension.
 27. The rechargeable persistentluminescence nanocomposites of claim 18, wherein the NIR-emitting isdetectable at least 10 hours after excitation.